SUMMARY Hematopoietic stem cells (HSCs) supply the lifelong foundation of the blood and immune systems. HSCs are therapeutically valuable as HSC transplantation is the standard of care for many hematological diseases. However, treatment availability remains problematic due to immune incompatibility and donor shortage. Likewise, while the number of transplanted HSCs directly impacts outcome, there are currently no established clinical protocols to successfully expand donor-harvested HSCs, nor to differentiate embryonic or induced pluripotent stem cells (iPSCs) into functional HSCs in vitro. Therefore, the identification of novel modifiers of de novo production of HSCs with long-term self-renewal and differentiation capacity is a major unmet clinical need. Despite decades of research, current protocols rely primarily on transcription factor overexpression of force cells into an ?HSC-like? program; however, transplantation of these in vitro-derived HSCs yields only limited long-term engraftment and multilineage potential. These observations imply that current in vitro differentiation strategies are missing critical cues which are essential to unlock or maintain full HSC function in vivo. HSCs are first formed in the vertebrate embryo from a unique population of mesodermal precursors termed hemogenic endothelium through a highly conserved process known as endothelial-to-hematopoietic transition. We have previously utilized the zebrafish model to discover novel regulators of HSC formation, including the first therapeutic identified in zebrafish to be used in FDA-approved clinical trials. In addition, we have demonstrated that key extrinsic signals occurring during embryogenesis coordinate the timing and scale of HSC production. More recently, our lab revealed an essential role for inflammatory signaling in developmental HSPC formation. However, the mechanism by which sterile inflammation originates in the embryo has not been determined. Our preliminary data indicate that physiological changes in metabolic state during embryogenesis initiate sterile inflammatory signaling to stimulate HSC commitment and expansion, without loss of multi-potency. Our central hypothesis is that ?developmental stressors?, such as onset of metabolic activity, activate the NLRP3-inflammasome to drive sterile IL1?-mediated commitment to HSC production. Our proposed work will demonstrate the essential role of developmental inflammasome activation in establishing HSPC commitment and expansion in vivo in zebrafish embryos and in vitro in human iPSC culture. Defining the molecular signaling pathways that mediate productive HSC formation in vivo will reveal new targets for optimizing the directed expansion and/or de novo production of human HSCs for therapeutic use.